During the past years, infectious diseases have increased and multi-drug resistance microorganisms have arisen (Acar J and Courvalein P pp 50-53; Aubry-Damon H and Andremot A, pp 54-55; Trieu-Cout P and Poyart C, pp 62-66, in ‘La Recherche’, vol 314, 1998).
Outbreaks of infectious diseases have generated the necessity of typing the causative microorganisms. Typing is the process by which different species of microorganisms of a given genus are classified in different subgroups or subtypes (Busch U and Nitschko H, J Chromatogr B, 1999, 722:263-278). Typing is important, from the epidemiological point of view, for recognizing outbreaks of infection, determining the way of transmission of nosocomial pathogens in the health centers and detecting the sources of the infections. It is also useful for identifying new virulent strains and monitoring vaccination programs. A typing process is been considered adequate if it fulfill the following criteria (Maslow J N et al., Clin Infect Dis, 1993, 17:153-164):                1. To give unambiguous results for each isolate analyzed.        2. To give reproducible results.        3. To differentiate unrelated strains within specie.        
There are several microorganism typing methods. Some of them are based on the analysis of phenotypic features (phenotypic methods) and others on the analysis of genotypic features (genotypic methods). Phenotypic methods detect features expressed by microorganisms, whereas the genotypic ones evidence the differences among the DNA of microorganisms. Thus, phenotypic methods have the disadvantage of giving an indirect measure of the changes in the genetic background. Said disadvantage does not occur with the use of genotypic methods.
One of the genotypic typing methods most widely used is Pulsed Field Gel Electrophoresis (PFGE). This method is considered the gold standard for the molecular typing of microorganisms. PFGE typing is performed by separating in gels DNA molecules that are subjected to the action of electric pulses in two different directions. After electrophoresis, the band patterns given by DNA molecules of an isolate of microorganism are highly reproducible and discriminatory and characterize unequivocally its DNA (Oliver D M and Bean P, J Clin Microbiol, 1999, 37(6):1661-1669). Additionally, the whole genomes of numerous isolates can be compared in a single gel. Thus, PFGE has been proposed as the optimal typing method (Maslow J N, Mulligan M E and Arbeit R D, Clin Infect Dis, 1993(17):153-164; Busch U and Nitschko H, J Chromatogr B, 1999(722):263-278). The results obtained by PFGE depend on the experimental conditions applied in DNA separation and on the genus and specie of the microorganism subjected to study. Thus,    a) If the microorganism has several lineal chromosomes of sizes lower than 10 Mb (1 megabase=1 000 000 base pairs) the band pattern given by its chromosomes is obtained. Said band pattern is called ‘the electrophoretic karyotype’. For instances, the microorganism can be yeast, unicellular parasites, etc.    b) If the microorganism has a single large circular chromosome the band pattern given by the macrorestriction fragments of said circular DNA is obtained. These patterns are called pulsetypes, since they are obtained in specific experimental conditions. For instances, the microorganism can be bacteria such as Escherichia coli, Staphylococcus aureus, etc.
The comparison of the electrophoretic karyotypes of different strains permits their characterization and differentiation. The same occurs with the restriction fragments of the bacterial DNA. Thus, both, the molecular karyotypes and the pulsotypes are used in the comparative study of fungi, bacteria and parasites. The routinely use of PFGE in medical microbiology has generated the necessity of improving the methods of sample preparation.
It also generated the need of designing a priori the running conditions to adequately separate the molecules and for analyzing the resulting electrophoresis patterns.
In general, the process of microorganism typing by PFGE comprises the following steps and procedures:    1) Preparing the samples: growing the microorganisms in nutrient broth, embedding the cells in gel and obtaining immobilized and deproteinized intact DNA molecules.    2) Designing the electrophoresis run: selecting the experimental conditions that should be set in PFGE equipments to obtain the optimal separation between the molecules.    3) Loading the samples in the gels and performing pulsed field gel electrophoresis to separate the DNA molecules.    4) Analyzing the band patterns obtained after the electrophoresis and comparing the results given by different isolates of microorganisms.
The equipments and procedures currently used in microorganism typing by PFGE are analyzed.
Pulsed Field Gel Electrophoresis
Pulsed field gel electrophoresis (PFGE) dates from 1984, when Schwartz and Cantor (Cell, 37, 67-75, 1984; U.S. Pat. No. 4,473,452) observed that applying electric pulses that periodically switched their orientation by a certain angle in relation to the agarose gel, large intact DNA molecules were resolved as band patterns.
The authors also determined that the separations of the molecules essentially depend on the duration of the electric pulses. Later, the angle formed by the field force lines, the electric field strength, the experimental temperature, the ionic strength of the buffer solution, the concentration of the agarose gel and the thickness of the agarose plugs, where the samples are embedded, were determined as the most important factors that influenced the resolution of DNA molecules. (Birren B. and Lai E. Pulsed Field Gel Electrophoresis: a practical guide. Academic Press. New York, 1993, p 107, 111, 129, 131, 135; López-Cánovas L. et al., J. of Chromatogr. A, 1998, 806, 123-139; López-Cánovas L. et al., J. of Chromatogr. A, 1998, 806, 187-197).
Different systems to perform PFGE have been developed. They have characteristic chambers with electrodes placed in different arrangements. Among these chambers are the OFAGE (Orthogonal Fi Id Alternating Gel Electrophor sis, Carle C. F. and Olson M. V. Nucleic Acids Res. 1984, 12, 5647-5664) CHEF (‘Contour Clamped Homogeneous Electric Field’, Chu G. Science 234, 1986, 1582-1585), TAFE (‘Transversal Alternating Field Electrophoresis’, U.S. Pat. No. 4,740,283), FIGE (‘Field Inversion Gel Electrophor sis’, U.S. Pat. No. 4,737,251 of Carle G. F. and Olson M. V) arrangements of electrodes, and the minichambers MiniTAFE and MiniCHEF (Riverón, A. M. et al., Anal. Lett, 1995, 28, 1973-1991; European Patent Application EP 0 745 844, Bula. 1996/49; U.S. patent application Ser. No. 08/688,607, 1995; Cuban patent RPI Nro. 02/95, 1995).
Commonly Used Systems for Microorganism Typing by PFGE
The most used systems for microorganism typing based on DNA analysis by PFGE are CHEF and TAFE. They provide straight band patterns in every lane of the gel, and thus, allow the comparison of the results obtained in a single run or in different electrophoresis runs.
The electrodes of CHEF system are placed in a hexagonal array around the gel and the voltages are clamped in them to guarantee homogenous electric field throughout the gel. The generation of homogeneous electric field throughout the chamber permits to obtain straight bands and reproducible migrations in the lanes of the gel. The electrodes of opposed polarities are 33.5 cm separated in CHEF chambers. It uses submarine gels that can be up to 21×14 cm in width and length. The gels are placed horizontally (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide. Catalog Numbers 170-3670 to 170-3673, BioRad, pp 11, 1995). As mentioned, CHEF systems have been extensively used for microorganism typing. For instances, bacteria (Beverly, S, Anal Biochem, 1989, 177:110-114; Dingwall A and Shapiro L, Proc Natl Acad Sci USA, 1989, 86:119-123; Ferdows M S and Barbour A G, Proc Natl Acad Sci USA, 1989, 86:5960-5973; Kohara Y, Akiyma K and Isono K, Cell, 1987, 50:495-508; Ohki M and Smith Cl, Nucleic Acids Res, 1989, 17:3479-3490; Schoenline P V, Gallman L M and Ely B, Gene, 1989, 70:321-329; Ventra L and Weiss A S, Gene, 1989, 78:29-36), Pseudomonas (Bautsch W, Grothues D and Tummler B, FEMS Microbiol Lett, 1988, 52:255-258; Romling U and Tummler B, J Clin Microbiol, 2000, 38(1):464-465), S. cerevisiae (Albig W and Entian K D, Gene, 1988, 73:141-152; Zerva L et al., J Clin Microbiol, 1996, 34(12):3031-3034), E. histolytica (Petter R et al., Infect Immun, 1993, 61(8):3574-3577), S. pneumoniae (McEllistrem M C et al., J Clin Microbiol, 2000, 38(1):351-353), S. aureus (Wichelhaus T A et al., J Clin Microbiol, 1999, 37(3):690-693), M. tuberculosis (Singh S P t al., J Clin Microbiol, 1999, 37(6):1927-1931), P. haemolytica (Kodjo A et al., J Clin Microbiol, 1999, 37(2):380-385), etc.
Due to the large dimensions of the CHEF chamber, it requires large amount of buffer solution to cover its electrode platform. Thus, the current intensity in the chamber can reach high values, even when low electric fields intensities are applied. Therefore, CHEF experiments demand power supplies of high rated power output. Besides it, large amount of heat is generated in the chambers, avoiding the reduction of run duration by increasing the electric field. CHEF chambers need at least 20 hours of electrophoresis to separate Saccharomyces cerevisiae chromosomes (molecules smaller than 1.6 Mb. 1 Mb=106 base pairs) in the characteristic pattern of 11 bands, and to type different strains of this yeast (Zerva L, et al., J Clin Microbiol, 1996, 34(12): 3031-3034). The CHEF chamber takes long time for separating the macrorestriction fragments of bacterial DNA molecules, since 20 hours, or more, are needed (van Belkum A et al., J Clin Microbiol 1998, 36(6):1653-1659; Marchadin H et al., Antimicrob Agents Chemother, 2000, 44(1):213-216; Romling U and Tummler B, J Clin Microbiol, 2000, 38(1):464-465). Similarly, long running times are needed to study parasites such as Entamoeba histolytica. To separate its chromosomes 24 hours are needed, at least (Petter R et al., Infect Immun, 1993, 61(8):3574-3577).
The advantage of currently used CHEF equipments is the possibility of analyzing 40 samples in a single run. This high throughput sample format facilitates the comparative analysis of the electrophoresis patterns given by samples of numerous isolates.
The TAFE system was proposed by K J Gardiner, W Laas and D Patterson in the paper published in Somatic Cell Mol Genet, 1986(12): 185. They called initially the system as “Vertical Pulsed Field Electrophoresis” (VPFE) and developed the equipment that was protected by the U.S. Pat. No. 4,740,283 of Apr. 26, 1988.
In TAFE system, two electrode pairs are placed parallel to both faces of the submarine gel (10×7.6×0.64 cm, length×width×thick), that is placed vertically in the chamber. Said electrode disposition generates electric field force lines that cross transversally the gel and compels the molecules to migrate through the gel thickness during each pulse. In TAFE, homogeneously sized molecules travel similar distances and migrate up to the same height in the gel leaving straight tracks, regardless the positions of the wells (into which the samples were loaded) in the gel. Thus, this system is useful for microorganism typing, since it facilitates the comparative analysis of the electrophoresis patterns given by samples of numerous isolates.
Based on these principles, Beckman Instruments manufactured the equipment called “Geneline I, or Transverse Alternating Field Electrophoresis System” (Beckman, The Geneline System Instruction Manual, ed. Spinco Division of Beckman Instruments Inc., 1988), which is also known as TAFE. This system uses a gel (11×7.2×0.6 cm, length×width×thickness) into which 20 samples can be loaded. Nevertheless, TAFE also requires large amount of buffer solution (3.5 liters) and biological sample. The equipment demands, at least, 20 hours to resolve the E. histolytica chromosomes (Báez-Camargo M et al., Mol Gen Genet 1996, 253:289-296). So, TAFE share the drawbacks with CHEF in microorganism typing.
As a conclusion, the commercially available equipments most frequently used to characterize the genome of microorganisms and parasites require long running time, and large amount of reagents and biological samples to resolve large DNA molecules in their band pattern.
FIGE system has been used for rapid typing of bacteria (Goering R V and Winters M A, J Clin Microbiol, 30(3):577-580, 1992; Grothues D et al., J Clin Microbiol, 26(10):1973-19771988). However, electrophoretic mobility inversion of DNA has been documented in FIGE experiments. Mobility inversion of DNA prevents correct size estimations and makes difficult the interpretation and comparison of the patterns given by numerous samples. The impossibility of predicting the moment of occurrence of DNA mobility inversion is one of the problems associated with said phenomenon (Birren B and Lai E. Pulsed Field Gel Electrophoresis: a practical guide, pp 10, Academic Press, Inc. San Diego, Calif. 1993). The main disadvantage of mobility inversion is the impossibility of identifying unambiguously the molecules present in a given band. To do it, the bands should be transferred and hybridized with specific probes. Hybridization notably increases the prices of the assay and is time demanding, thus the typing process will be more expensive and time consuming.
The attempts to reduce the electrophoresis time in current CHEF equipments, such as GeneNavigator (Amersham-Pharmacia-LKB, Pharmacia Molecular and Cell Biology Catalogue, Pulsed Field Gel Electrophoresis, Nucleic Acids Electrophoresis. 1998, pp 77-79), CHEF DRII and CHEF Mapper (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide. Catalog Numbers 170-3670 to 170-3673. Bio-Rad, pp 11, 1995) by increasing the voltage applied to the electrodes is nearly impossible. It is due to the limit of power supply output and the cooling system. Consequently, manufacturers recommend 9 V/cm as maximum electric field to apply in said equipments (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide. Catalog Numbers 170-3670 to 170-3673. Bio-Rad, pp 2, 1995). Therefore, the reduction of the electrophoresis duration by increasing the electric field intensity is prevented in CHEF system. Current TAFE equipments have similar problems.
PFGE Miniequipments
MiniPFGE equipments, miniCHEF and miniTAFE versions, were reported in 1995 (Riverón A M et al., Anal. Lett., 1995, vol. 28, pp 1973-1991; European Patent Application EP 0 745 844, Bull. 1996/49; U.S. patent application Ser. No. 08/688,607, 1995; Cuban Patent RPI 02/95, 1995). Miniequipments overcome most of the mentioned drawbacks of the PFGE equipments. Pulsed field electrophoresis experiments are performed in a minigel (4×4×0.5 cm; length×width×thickness) loaded with 7 samples in 4 to 6 hours. Electric field strength reaching 16 V/cm can be applied in MiniCHEF, providing good resolution of the electrophoresis band patterns in 2.7 hours. The short distance between the electrodes of opposed polarities permits to construct small chambers and to use small amount of buffer volumes to cover the electrodes. By applying a given voltage to a miniCHEF and CHEF chambers (certain electric field strength value ‘E’) the heat generated in the minichamber is always lesser than the heat generated in the commercially available CHEF (Riverón A M et al., Anal. Lett., 1995 vol. 28, pp. 1973-1991).
MiniTAFE equipments admit 22 V/cm, achieving resolution among the bands (Riverón A M et al., Anal. Lett., 1995 vol. 28, pp. 1973-1991). MiniTAFE permits to obtain the S. cerevisiae electrophoretic karyotype in 5 hours. MiniTAFE chambers with short separation (7.8 cm) between opposite electrodes are small and use small amount of buffer solution. However, if samples thicker than 0.1 cm are loaded in the minigels, longer running times are needed to achieve good resolution of the electrophoresis patterns. According to previous reports, sample thickness influences electrophoresis running time (López-Cánovas et al., J. Chromatogr. A, 1998, 806, pp. 187-197). As the samples are thicker, longer gels are needed to obtain the same band patterns. However, the reported miniCHEF minigels admit only 7 samples, whereas the miniTAFE supports 13 samples. They are low throughput sample formats for typing isolates of microorganisms in clinical laboratories.
Despite miniPFGE equipments have advantages over currently used systems, neither miniCHEF nor MiniTAFE were used for microorganism typing. Maybe, it obeys to the attempting of using samples as thick as the ones used in conventional gels. In addition, simple procedures to select miniequipment running parameters are not available.
Preparation of Immobilized DNA
A method to prepare intact DNA molecules is essential for microorganism typing by PFGE in current equipments or miniequipments. Previously reported methods of DNA isolation and purification in solution provoke shearing of said molecules (Schwartz D C and Cantor C R, Cell, 1984, 37, pp. 66-75). Schwartz and Cantor proposed a methodology to prepare samples for PFGE and only excluded the molecules with sizes smaller than 1 Mb (106 base pairs). The methodology consists in harvesting the cultured cells, washing the cells and embedding them in agarose plugs. In ulterior steps, spheroplasts (if cellular wall exists) are formed ‘in situ’ and further lysed in said plugs. Finally, the immobilized DNA molecules are deproteinized using proteinase K. The method has been effective to prepare samples from microorganisms of different genus, species and origins. However, spheroplasts need to be formed if said microorganisms possess cell wall, and the enzymes needed to form spheroplasts, as well as the proteases, are expensive. The reported procedure requires that samples were incubated overnight twice, which is 32 hours for sample preparation (U.S. Pat. No. 4,473,452, Sep. 25, 1984).
More recently, Gardner (Gardner D C J et al., Yeast, 1993, 9, 1053-1055) obtained band patterns of S. cerevisiae chromosomes from cells that did not form spheroplasts. In parallel, Higginson et al (Higginson D. et al., Anal. Lett., 1994, 27:7, 1255-1264) showed that S. cerevisiae DNA can be deproteinized using 8 M urea instead of proteinase K. However, the method described by Gardner is still expensive, because he used proteases to deproteinize the DNA, whereas, the method described by Higginson is cheap, but consumes 72 hours of incubation, which is a long time. Later, S. cerevisiae samples were prepared, and enzymes were not used: The plugs were sequentially incubated with LETK (10 mM Tris, 500 mM EDTA, 600 mM KCl, pH 7.5) for 4 hours, NDS (10 mM Tris, 500 mM EDTA, 1% sarcosyl, pH 9.5) for 2 hours, and NDS plus 4 M urea (NDSU) for 2 hours. This method needed 10 hours for sample preparation (López-Cánovas L, et al., Anal. Lett, 1996, 29:12, 2079-2084). Rapid methods for the preparation of immobilized DNA were described also, but they use enzymes (for instance, in Guidet F and Langridge P, Biotech, 1992, 12:2, 222-223).
As general rule, immobilized DNA for PFGE experiments requires the formation of spheroplasts and DNA deproteinization using proteases. These requirements are independent from the cell type studied (bacteria, yeast, etc) (Maule J, Mol. Biotech. 1998, 9: 107-126; Olive D M and Bean P, J. Clin. Microbiol, 1996, 37:6, 1661-1669). For instances, immobilized Staphilococcus aureus DNA were reported to be prepared in 2 hours by immobilizing cells with lysostaphin and incubating the plugs with detergents, whereas Streptococcus fecalis cells needed to be incubated with lysozyme and mutanolysin prior to the immobilization (Goering R V. and Winter M A., J. Clin. Microbiol, 1992, 30:3, 577-580). In the mentioned work, authors did not incubate the samples with proteinase K. However, Matushek et al. (Matushek M G et al., J. Clin. Microbiol, 1996, 34:10, 2598-2600) reported that they were unable to obtain the band patterns if the samples were not incubated with proteinase k. As alternative, they proposed a rapid method to prepare immobilized DNA samples using proteinase K. Recently, McEllistrem et al. (McEllistrem M C et al., J. Clin. Microbiol, 2000, 38:1, 351-353) reported a complete non-enzymatic method to prepare immobilized DNA of Streptococcus pneumoniae. However, it consumes 6 hours and the authors attributed the protocol success to the activation of an autolysin of the Streptococcus. At present, the spheroplast formation enzymes and the proteases have been deleted only from the protocols of preparation of intact DNA molecules of S. cerevisiae and Streptococcus pneumoniae, but the times required to complete these preparations are 10 and 6 hours, respectively, which are long times. Consensus does not exist about the feasibility of deleting the protoplast formation enzymes and proteinase K in sample preparation of microorganisms. Therefore, current methods are expensive and consume long times.
Most of the above methods rest on the assumption that microorganisms with cell walls must be immobilized previously to the enzymatic treatment. An exception is presented in the paper published by Goering and Winter (Goering R V and Winter M A, J. Clin. Microbiol, 1992, 30:3, 577-580). The authors suspended cells in a solution containing lysozyme and mutanolysin (two enzymes to form spheroplast) prior to their immobilization. However, a general non-enzymatic method to treat microorganisms with cell walls prior to their embedding in agarose gels has not been presented yet. Such method would be cheaper and simpler that current protocols in the preparation of samples.
Methods to isolate nuclei acids in solution, starting from cells heated in the presence of agents that provoke the permeability of the microorganism cell wall, were reported (European Patent 0,657,530, 1994, bulletin 95/24; U.S. Pat. No. 158,940, 1993). The method releases large fragments of undegraded nucleic acids from microorganisms without physically disrupting the entire cell wall. This method does not require lytic enzymes. However, intact DNA molecules are not obtained, because DNA is isolated in solution; and the authors did not propose the method to obtain the molecular karyotypes or the pulsetypes of the mentioned microorganisms. Authors reported that the obtained DNA is suitable for hybridization, but they did not address the possibility of DNA restriction with endonucleases. In conclusion, said method does not guarantee the obtaining of intact DNA molecules, whereas the possibility of digesting them with restriction enzymes remains unknown.
Therefore, a general procedure has not been proposed yet for the preparation of immobilized intact DNA from yeast, gram-positive and gram-negative bacteria and parasites by non-enzymatic methods in short times.
The preparation of immobilized DNA samples needs molds to form said samples. These molds can be reusable or disposable. The reusable molds should allow the sterilization. This is important for handling samples from pathogenic microorganism. The use of disposable molds requires continuous supply, which can be a limiting factor in laboratories with low budget.
Known molds are the following:    a) Molds that form independent and similar plugs (U.S. Pat. No. 4,473,452, Sep. 25, 1984);    b) Molds that form long ribbons, which are cut to form independent plugs;    c) Molds that form long square ‘noodle’ or long agarose rods, which are cut to form independent plugs (Birren B and Lai E. Pulsed Field Gel Electrophoresis: a practical guide, Academic Press, Inc. San Diego, Calif., 1993, pp 29-30).
In general, samples of dimensions larger than the gel slot are generated in said molds. For this reason, to obtain plugs with dimensions similar to the ones of slots, the ribbons, noodle, etc, need to be cut with a razor blade or another device. (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide pp 40, Section 7. Catalog Numbers 170-3670 to 170-3673. Bio-Rad. 1995). However, cutting the ribbons or noodles provokes plugs of non-homogeneous sizes and dimensions, which influence the quality of the electrophoresis patterns. DNA molecule resolutions in the gel depend on the sample thickness. Consequently, the comparison of the patterns obtained in different lanes of the gel is difficult. Difficulties in the comparisons of band patterns represent a disadvantage in microorganism typing.
A mold for embedding cells in agarose gels and treat plugs was disclosed in the U.S. Pat. No. 5,457,050, Oct. 10, 1995. The mold could be disposable or reusable, depending on the material used to make it. It is claimed that the facility of the mold is that samples are formed and treated inside said mold. However, it is a disadvantage: if the plugs are kept inside the mold during the incubations, the time needed to obtain the samples suitable for PFGE analysis is notably lengthened. The effector molecules of the lysis and deproteinization solutions can poorly reach the target molecules inside the cells, because the contact area between the plugs and the incubation solutions is at least reduced to half.
Selection of the PFGE Experimental Conditions
The selection of the experimental conditions is complex. Methods to select the running PFGE conditions have been reported.
For example, the CHEF Mapper from Bio-Rad has an option of auto-algorithm and another one of interactive algorithm (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide. pp 31-40 Catalog Numbers 170-3670 to 170-3673. Bio-Rad. 1995). Both options permit to calculate the pulse times, pulse ramp durations, reorientation angle, electric field and optimal running time to separate DNA molecules of a given sample. In contrast to the auto-algorithm, in which fixed experimental conditions are assumed, the buffer type, temperature and concentration, and the agarose type and concentration are permitted to vary as inputs in the interactive algorithm. Both algorithms work based on empirical and theoretical data collected during 5 years of experience (Bio-Rad Catalogue. Life Science Research Products. Bio-Rad Laboratories, pp185. 1998/99). However, the manufacturers recommend feed the auto-algorithm with DNA sizes smaller and larger than the expected sizes of the sample molecules. In addition, if extremely wide size ranges are entered to the auto-algorithm, as well as to the interactive program, erroneous results, such as band inversion in the mid range of the gel, can be generated.
Another empirical expression was proposed to give the electric pulse duration that separates the group of molecules of sizes falling between a given value and a higher one called RSL (Resolution Size Limit) (Smith D R. Methods I, 1990, 195-203). However, said expression is valid on specific experimental conditions alone, and it does not predict the resolution between two any molecules. Another function was proposed. It provides the approximate conditions of electric field and pulse time needed to separate a given group of molecules (Gunderson K and Chu G, Mol. Cell. Biol., 1991, 11:3348-3354). However, said function only permits to obtain rough estimates of the two mentioned variables, and does not provide the molecule migrations at any experimental condition.
Despite various theoretical studies were performed about DNA molecular reorientation during PFGE (Noolandi J, Adv. Electrophoresis, 1992, 5: 1-57; Maule J, Mol. Biotech. 1998, 9:107-126), said studies have not given a practical and useful result in the laboratories yet. They did not generate methods allowing the PFGE user to select and set the experimental conditions needed to separate the molecules under analysis.
The equations proposed by López-Cánovas et al. (López-Cánovas L et al., J. Chromatogr. A, 1998, 806:123-139) to describe DNA migration in PFGE, have not been used to predict the band patterns that should be obtained when varying the pulse time ramps, the electric field, temperature and running time. These variables are usually modified in microorganism typing.
PFGE Band Pattern Analysis
Computerized systems for image acquisition data from PFGE gels are available for band pattern analysis (Gerner-Smidt P et al., J. Clin. Microbiol, 1998, 37(3):876-877; Tenover F et al., J. Clin. Microbiol, 1995, 33(9):2233-2239; van Belkum A et al., J. Clin. Microbiol, 1998, 36(6):1653-1659). However, the comparison of the restriction patterns remains a subjective process, and it cannot be totally reduced to rigid algorithms (Tenover F et al., J. Clin. Microbiol, 1995, 33(9):2233-2239). Although computer based analyses were performed, the final interpretation of the patterns must be subordinated to previous visual inspection.
Automatic and non-automatic band pattern analysis give as result the number of bands and the sizes of the molecules in the bands. It is usually done by comparing unknown DNA migrations with the migrations of molecular weight markers. However, PFGE is relatively new, and size markers for wide DNA size range are not always available. Consequently, the criteria used for bacterial typing, based on the interpretation of PFGE band patterns, consist in the determination of the number of different restriction fragments found when digesting the DNA of the microorganisms under comparison.
Equations to describe DNA molecule migrations under a single pulse ramp, different electric field strength and distinct temperature were proposed from electrophoresis data collected in experiments done in 1.5% agarose, Lachema and 0.5×TBE, 1×TBE: 89 mM Tris, 89 mM Boric Acid, 2 mM EDTA (López-Cánovas L et al., J. Chromatogr. A, 1998, 806:123-139). However, a method to extend and apply said equations to the analysis of band patterns after the application of pulse time ramps does not exist. The application of pulse time ramps is usually done in the comparative study of microorganisms.
Current Process for Microorganism Typing by PFGE
Total process for microorganism typing needs long time and many resources. Electrophoresis demands around 20 hours, methods of sample preparation, recommended by manufacturers or reported in the literature, require to use large amounts of enzymes (For instances, 80 mg/ml proteinase k) and long incubation times (CHEF Mapper XA Pulsed Field Electrophoresis System. Instruction Manual and Application Guide. pp 40-43 Catalog Numbers 170-3670 to 170-3673. Bio-Rad. 1995). A factor limiting the use of PFGE for microorganism typing is the time needed to complete the analysis of isolates, It is from 2 to 3 days, thus reducing the capacity of the laboratories to analyze many samples (Olive D M and Bean P, J. Clin. Microbiol, 1999, 37:6, 1661-1669).